Selective Detection and Assignment of the Solution NMR Signals of

Mitsuo Umetsu , Tomoyuki Kadota , Zheng-Yu Wang , Yoshikazu Tanaka , Tadafumi Adschiri , Tsunenori Nozawa. Chemistry Letters 2005 34 (7), 940-941 ...
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Selective Detection and Assignment of the Solution NMR Signals of Bacteriochlorophyll a in a Reconstituted Subunit of a Light-Harvesting Complex Zheng-Yu Wang,* Yoshiyuki Muraoka, Masahiro Shimonaga, Masayuki Kobayashi, and Tsunenori Nozawa Contribution from the Department of Biomolecular Engineering, Faculty of Engineering, Center for Interdisciplinary Research, Tohoku UniVersity, Sendai 980-8579, Japan Received May 29, 2001

Abstract: High-resolution solution NMR spectra have been obtained for bacteriochlorophyll (BChl) a molecules in a biologically functional subunit of a bacterial core light-harvesting complex based on a modified reconstitution method. The reconstituted subunit of pigment-integral membrane polypeptides is stable and homogeneous at high concentrations at room temperature and exhibits a Qy absorption peak at 818 nm. 1H and 13C chemical shifts have been specifically assigned for BChl a using the fully and selectively 13Clabeled pigments incorporated with natural abundance polypeptides in deuterated detergent solution. Remarkable signal broadening has been observed upon reconstitution, where the bacteriochlorin macrocycle is shown in a highly restricted molecular motion while the phytol side chain remains relatively mobile. Two sets of resonances are revealed for 32, 81, 10, 121, and 134 protons, and 82 methyl protons exhibit four resonances with large upfield complexation shifts. The result indicates a nonequivalent state for the two BChl a molecules in the subunit and can be best interpreted in terms of a parallel face-to-face configuration with partial overlap over the pyrrolic rings II, III, and V. In comparison with BChl a in acetone, 82, 132, and 134 protons are largely perturbed, and the propionic and phytol side chain may adopt a different conformation in the reconstituted subunit. The 13C chemical shift of 31 carbonyl carbon shows a large change downfield, indicating strong hydrogen bonding for all the acetyl carbonyls. Carbonyl carbons at 131 give rise to two 13C resonances with equal intensities, suggesting that the keto carbonyl in one BChl a molecule within a subunit forms a stronger hydrogen bond than that in another BChl a molecule.

Introduction

Despite intense interest and continuing effort, the molecular organization of bacteriochlorophyll (BChl) in the core lightharvesting complex (LH1) of purple photosynthetic bacteria has not been determined to atomic resolution. To date, the highestresolution structure of LH1 is obtained as a 2D projection map at 8.5 Å produced by electron diffraction using 2D crystals of reconstituted LH1 complexes from a purple nonsulfur bacterium Rhodospirillum (R.) rubrum. In contrast, a number of highresolution X-ray crystal structures have been determined for the reaction center (RC)1-3 and peripheral light-harvesting complex (LH2).4,5 The difficulty may be partially due to the unique in vivo location of LH1, where the integral membrane pigmentprotein complex is sandwiched between the RC and LH2 components in most species, making it difficult to isolate intact (1) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618-624. (2) Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730-5734. (3) Nogi, T.; Fathir, I.; Kobayashi, M.; Nozawa, T.; Miki, K. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13561-13566. (4) McDermott, G.; Prince, D. M.; Freer, A. A.; Hawthornthwaite-Lawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaac, N. W. Nature 1995, 374, 517521. (5) Koepke, J.; Hu, X.; Muenke, C.; Schulten, K.; Michel, H. Structure 1996, 4, 581-597. 1072 VOL. 124, NO. 6, 2002

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LH1 complex and to prepare high-quality crystals. Hitherto, a large body of biochemical and spectroscopic data has been collected to provide information on the structure and function of the LH1 complex.6,7 A resonance Raman study has utilized the carbonyl stretch region (1600-1700 cm-1) to probe the interaction mode of carbonyl groups of the BChl a molecule with its polypeptide environment and revealed that acetyl carbonyl groups are hydrogen bonded in its native form.8 At present, only a reconstitution methodology using various BChl analogues has been employed to examine the structural requirements of the BChl binding in the LH1 complex.9 However, the molecular basis is not fully understood for the role of each individual functional group of BChl a. We describe here the first observation of high-resolution solution NMR signals from intact BChl a molecules in a reconstituted subunit of the LH1 complex and show several remarkable characteristic features of the complexation-induced chemical shift and intensity (6) Loach, P. A.; Parkes-Loach, P. S. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. D., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995; pp 437-471. (7) Cogdell, R. J.; Isaac, N. W.; Howard, T. D.; McLuskey, K.; Fraser, N. J.; Prince, S. M. J. Bacteriol. 1999, 181, 3869-3879. (8) Visschers, R. W.; van Grondelle, R.; Robert, B. Biochim. Biophys. Acta 1993, 1183, 369-373. (9) Davis, C. M.; Parkes-Loach, P. S.; Cook, C. K.; Meadows, K. A.; Bandilla, M.; Scheer, H.; Loach, P. A. Biochemistry 1996, 35, 3072-3084. 10.1021/ja0112994 CCC: $22.00 © 2002 American Chemical Society

Solution NMR of BChl a in B820 Complex

changes on the basis of the assigned resonances. This work provides complementary information to the ongoing effort of crystal structure determination of LH1, in terms of pigmentpigment and pigment-protein interactions. The system of this study, as a model of chromophore-protein complexes, also has potential applications in the peripheral areas, such as heme proteins and another type of photoreceptors: rhodopsin/bacteriorhodopsin. LH1 of photosynthetic bacteria generally comprises two small polypeptides, R and β, with a ratio of 1:1, along with two BChl and one or two carotenoid molecules per Rβ pair. The R- and β-polypeptides used in this study were obtained from wild-type R. rubrum. The BChl a is believed to be ligated by a histidine residue to its central Mg atom and absorbs at 880 nm (Qy band) in the near-infrared region for the native LH1 of R. rubrum, a substantial red shift (110 nm) compared to the absorption of its monomeric form in organic solvents. The LH1 complex is capable of forming a stable intermediate species, characterized by a Qy absorption at approximately 820 nm (referred to as B820), by dissociation of the LH1 with detergent n-octyl β-Dglucopyranoside (OG).10 The B820 species is considered to be a structural subunit of the LH1 complex and can be reassociated to form native LH1 as well as reversibly dissociated to its individual components by adjusting the detergent concentration. Furthermore, Parkes-Loach et al. have shown that both the B820 subunit and LH1 can be spontaneously reconstituted from the separately isolated polypeptides and BChl a.11 The selfassembling feature and relatively small size of the B820 subunit provide an ideal target for the structural analysis by NMR in the solution state. Attempts for NMR measurements of biologically active integral membrane pigment-protein complexes in solution state usually encounter at least three hurdles: (a) low spectral resolution due to the large molecular mass of the proteindetergent micelles, (b) poor selectivity in distinguishing the resonances between pigment molecule and protein, and (c) giant signals from the detergent molecules. Using an organic solvent system to solubilize the membrane protein is an alternative way to avoid the difficulties. However, this in most cases leads to a loss of functional property, implying a conformational change from its native form. Recently, Conroy et al.12 determined a solution structure of β-polypeptide of the LH1 by NMR. They used a chloroform/methanol mixed solvent system and found that BChl a is unable to bind to the polypeptide in this solvent system. Therefore, they were unable to observe structural changes on BChl ligation. We show in this study that the difficulties mentioned above can be overcome by choosing a pigment-protein complex with a minimum size while maintaining its functionality and by developing a method of preparing a highly concentrated sample suitable for the NMR measurement. In addition, we replaced the pigment molecules in the complex by various 13C-labeled equivalents, which give the same spectral features and significantly enhance the 1H-13C correlation signals. Furthermore, deuterated detergent has been used to suppress the strong resonances from the small molecules. (10) Miller, J. F.; Hinchigeri, S. B.; Parkes-Loach, P. S.; Callahan, P. M.; Sprinkle, J. R.; Riccobono, J. R.; Loach, P. A. Biochemistry 1987, 26, 5055-5062. (11) Parkes-Loach, P. S.; Sprinkle, J. R.; Loach, P. A. Biochemistry 1988, 27, 2718-2727. (12) Conroy, M. J.; Westerhuis, W. H. J.; Parkes-Loach, P. S.; Loach, P. A.; Hunter, C. N.; Williamson, M. P. J. Mol. Biol. 2000, 298, 83-94.

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Figure 1. Structure and nomenclature of BChl ap.

With all these efforts, we have succeeded in selectively detecting and specifically assigning the NMR resonances from the individual functional groups of BChl a molecule. Materials and Methods Preparation of 13C-Labeled BChl ap Molecules. Fully 13C-labeled BChl a containing phytol as the esterifying alcohol (BChl ap, Figure 1) was isolated from purple sulfur bacterium Chromatium Vinosum. The cells were grown in a medium containing NaH13CO3 (13C > 98% atom, Isotec Inc.) as the sole carbon source. Partially 13C-labeled BChl ap was isolated from purple sulfur bacterium Chromatium tepidum. The cells were grown in a medium containing NaHCO3 and sodium acetate as carbon sources. Three combinations of the carbon-containing chemicals were made in the preparation of culture media: (1) NaH13CO3 and CH3COONa, (2) NaHCO3 and 13CH3COONa (13C > 99%, Isotec Inc.), and (3) NaHCO3 and CH313COONa (13C > 99%, Isotec Inc.). All BChl ap molecules were extracted following procedures based on Omata and Murata13 and were purified on a reverse-phase HPLC column (ODS-80Ts, TOSOH) with solvent mixtures of acetonitrile/ acetone/methanol/water (33:50:15:2, volume ratio).14 Isolation and Purification of LH1 Apopolypeptides. The LH1 complex was isolated and purified in its native form from wild-type R. rubrum according to method B of Picorel et al.15 Fractions eluted from a DEAE column (25 mm × 250 mm, Toyopearl 650S, TOSOH) were monitored spectroscopically by measuring the absorbance between 250 and 900 nm. The ratios of absorbance at 880 nm (A880) to those at 800 nm (A800, due to the absorbance of RC) and at 280 nm (A280, due to the absorbance of aromatic amino acids) were used as indicators of purity. Complexes which gave ratios A880/A800 greater than 7.5 and A880/A280 greater than 2.6 were considered pure enough for use in the reconstitution experiment. After dialysis and drying, the purified LH1 was extracted repeatedly, first with benzene and then with methanol to remove carotenoids and BChl a. At this stage, a small amount of the apopolypeptides was chromatographed on a reverse-phase HPLC system (TSKgel, Super ODS, 4.6 mm × 100 mm, TOSOH) to further check the purity using a three-stage linear gradient.11 No other components other than the R- and β-polypeptides were detected.14 Reconstitution of B820 Subunit. Typically, 20 mg of lyophilized apopolypeptide was dissolved in 700 µL of phosphate buffer (D2O, 50 mM, pH 7.0) containing 5% deuterated OG (w/v, Anatrace Inc., D > 97%) and lyophilized after thorough mixing. Aliquots of 13C-BChl ap (13) Omata, T.; Murata, N. Photochem. Photobiol. 1980, 31, 183-185. (14) Wang, Z.-Y.; Shimonaga, M.; Muraoka, Y.; Kobayashi, M.; Nozawa, T. Eur. J. Biochem. 2001, 268, 3375-3382. (15) Picorel, R.; Belanger, G.; Gingras, G. Biochemistry 1983, 22, 2491-2497. J. AM. CHEM. SOC.

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Wang et al.

Figure 2. Absorption spectra of the reconstituted B820 complex (solid line) used for NMR measurements and BChl ap in acetone (dotted line) at room temperature. The spectrum of the highly concentrated B820 subunit solution was recorded using a cuvette with a light path length of 0.12 mm.

dissolved in acetone-d6 (D > 99.9%, Isotec Inc.) were added with a proportion of ODV770 ) 9.1 per mg of apopolypeptides and then freezedried after thorough mixing. A 700 µL volume of deuterated water (D > 99.9%, Isotec Inc.) was added to the pigment-polypeptide-detergent mixture and then was vortexed thoroughly. The solution was centrifuged at 14 000 g for 10 min. The supernatant was checked with absorption spectroscopy (Beckman DU-640) and used for NMR measurement. Normally, the precipitate contained about 5% of the original amount of polypeptides. All operations of the reconstitution were conducted in dim light at room temperature (∼22-23 °C). NMR Measurements. NMR spectra were collected on Bruker DRX400 and DRX-500 spectrometers at a temperature of 35 °C. One- and two-dimensional 1H-13C shift correlation spectra using 1H-detected heteronuclear multiple-quantum coherence via direct coupling method (HMQC) were acquired with the pulse sequence described by Bax et al.16 The spectral width for 1H was 4800 Hz and for 13C was 16 kHz. A total of 128 t1 points of 2K data points were acquired. For each t1 value, 320 or 400 transients were recorded, the number of scans depending on the experiment. During acquisition, the 13C spectrum was decoupled using a broadband GARP modulation.17 One-dimensional proton-decoupled 13C spectra were recorded with 30° pulse, 8K data points and 1.0 s repetition time. Refocused insensitive nuclei enhanced by polarization transfer (INEPT)18 spectra were recorded for the assignment of proton-bound carbons using selectively 13C-enriched samples. Chemical shifts were referenced to 2,2-dimethyl-2-silapentane5-sulfonate (DSS).

Results and Discussion

Overall Spectral Features. The reconstituted B820 subunit is fully functional even at high concentrations (∼1.8-2.2 mM protomer), as can be confirmed from the spectroscopic homogeneity of its absorption spectrum (Figure 2). The subunit is stable at room temperature for several days and exhibits a Qy band at 818 nm, whereas the BChl ap in acetone has a Qy absorbance at 770 nm. Figure 3 shows the two-dimensional 1H13C HMQC spectrum of the reconstituted B820 subunit incorporated with fully 13C-labeled BChl ap and solubilized in phosphate buffer containing 5% deuterated OG at 35 °C, together with that of the corresponding apopolypeptides under the same conditions. Significant signal broadening was observed for all protons and proton-attached carbons of the BChl ap (16) Bax, A.; Griffey, R. H.; Hawkins, B. L. J. Magn. Reson. 1983, 55, 301315. (17) Shaka, A. J.; Barker, P. B.; Freeman, R. J. Magn. Reson. 1985, 64, 547552. (18) Burum, D. P.; Ernst, R. R. J. Magn. Reson. 1980, 39, 163-168. 1074 J. AM. CHEM. SOC.

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molecule, indicating a largely reduced molecular motion upon introduction of the BChl ap into the polypeptide-detergent micelles. Despite the broadening, most resonances were resolved in the 2D spectrum. To facilitate assignment, similar measurements were performed on the reconstituted B820 complexes using selectively 13C-labeled BChl ap. Such experiments not only help distinguish the signals due to trace contaminants of small molecules but also improve the spectral resolution by reducing dipole-dipole couplings that may occur significantly for the B820 subunit reconstituted with fully 13C-labeled BChl ap. Using the results of selective labeling and comparing with the spectrum of intact BChl ap in acetone, we were able to specifically assign all the resonances of BChl ap in the B820 complex. One of the most significant features is that many proton resonances are multisplit with nonsymmetric spectral shapes. The result strongly suggests that BChl ap molecules in the B820 subunit exist in a nonequivalent configuration where the corresponding functional groups are surrounded by different local environments.19-21 The local effects may arise from the interaction with a neighboring BChl ap molecule or with surrounding amino acid residues and are consequently reflected in the chemical shift changes of the individual protons. Another characteristic feature observed in the NMR spectra is the differing dynamic behavior between the macrocycle and phytol side chain. The most obvious evidence can be found from comparison of the intensities between the meso CH and P2CH. While these methine groups showed similar intensities and line widths in the 1H and 13C spectra in acetone, considerably greater signal broadening was observed for the meso groups, particularly the protons, compared to that of P2 methine group (Figure 4). This corresponds to a much faster relaxation rate for the protons directly attached to the macrocycle and indicates that molecular motion of the macrocycle becomes highly restricted upon incorporation into the polypeptide environment, whereas the phytol chain remains in a relatively mobile mode as this portion is relatively distant from the Mg-His binding site. 1H NMR Assignment of the BChl a in Reconstituted B820 p Complex. Assignment of the 1H NMR spectrum for the BChl ap in the reconstituted B820 subunit largely relied on both the corresponding 1H and proton-attached 13C chemical shifts of BChl ap in organic solvents. Although the 1H chemical shifts of BChl ap have been established, these values vary with coordination and aggregation states in different solvents.22,23 Therefore, it was insufficient to use only the 1H chemical shift values obtained from organic solvents for the assignment of BChl ap in the reconstituted B820 complex, where the chemical shift changes induced by peptide binding were much greater than those observed from solvent effects. In contrast, complexation-induced 13C chemical shift changes are relatively small for the proton-attached carbons.19 This greatly assists the assignment of the 1H spectrum of BChl ap through H-C correlation. We have confirmed the assignment by 2D HMQC (19) Wang, Z.-Y.; Umetsu, M.; Kobayashi, M.; Nozawa, T. J. Am. Chem. Soc. 1999, 121, 9363-9369. (20) Wang, Z.-Y.; Umetsu, M.; Kobayashi, M.; Nozawa, T. J. Phys. Chem. B 1999, 103, 3742-3753. (21) Wright, K. A.; Boxer, S. G. Biochemistry 1981, 20, 7546-7556. (22) Brereton, R. G.; Sanders, J. K. M. J. Chem. Soc., Perkin Trans. 1 1983, 423-430. (23) Lo¨tjo¨nen, S.; Michalski, T. J.; Norris, J. R.; Hynninen, P. H. Magn. Reson. Chem. 1987, 25, 670-674.

Solution NMR of BChl a in B820 Complex

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Figure 3. Two-dimensional 1H-13C shift correlation spectrum of the reconstituted B820 subunit (∼2 mM protomers) using fully 13C-labeled BChl ap and natural abundance polypeptides solubilized in 5% deuterated OG-phosphate buffer (50 mM, D2O, pH 7.0) at 35 °C (red), together with that of the corresponding apopolypeptides (black) under the same conditions.

experiment using acetone-d6 as a solvent in which the BChl ap is present in a five-coordinate monomeric form.22 This spectrum (Supporting Information) was used as a reference in this study because BChl ap in the B820 complex is also believed to exist in a five-coordinated form. Table 1 shows the full assignment of 1H chemical shifts for the BChl ap in the reconstituted B820 subunit together with the complexation shifts defined by ∆δH ) δΗB820 - δΗ770(acetone-d6). Assignments of the meso protons, 5-, 20-, and 10-H, were straightforward, because these protons attach to the carbons whose chemical shifts in organic solvents have been well established.24-26 It was of interest to note that the chemical shift of 10-H for the BChl ap in B820 complex appeared at slightly higher field relative to that of 20-H, whereas the reverse is known for the BChl ap in organic solvents regardless of its coordination number and aggregation state.22 Assignments of 132-, P2-, and P1-H were also apparent, as their resonances were well separated from others in the 2D HMQC spectrum. These protons exhibited large downfield complexation shifts, up to 0.48 ppm, as shown in Figure 4. Four methine protons at 18, 7, 8, and 17 were assigned on the basis of 2D HMQC spectra using the selectively 13C-labeled BChl ap. Carbons at the 18 and 7 positions were found to be labeled in the medium containing CH313COONa, while carbons at the 8 and 17 positions were labeled in the medium containing 13CH3COONa. Methyl protons at 13,4 21, 121, and 32 were identified by their (24) Oh-hama, T.; Seto, H.; Miyachi, S. Arch. Biochem. Biophys. 1985, 237, 72-79. (25) Okazaki, T.; Kajiwara, M. Chem. Pharm. Bull. 1995, 43, 1311-1317. (26) Facelli, J. J. Phys. Chem. B 1998, 102, 2111-2116.

strong H-C correlation resonances together with the results of selective 13C-labeling experiments. While chemical shifts of the 21- and 121-methyl protons remained almost the same values as those in acetone, large upfield and downfield complexation shifts were observed for the 134- and 32-methyl protons, respectively. In the propionic side chain, resonance of 172 protons appeared at lower field relative to that of 171 protons, in reverse order for those of the BChl ap in acetone. Ethyl protons at 81 and 82 gave the largest upfield complexation shifts observed for the BChl ap in the reconstituted B820 complex. The 82-H resonances can be distinguished by H-C correlation, because 13C resonance of 82 carbon appears the most upfield. Because of structural similarity, phytol protons were less resolved on the 2D HMQC spectra except for P3a and P4 protons even using the selectively 13C-labeled BChl ap. Only P3a and P15 protons revealed relatively large downfield complexation shifts. Several peripheral groups of BChl ap in the reconstituted B820 subunit exhibit two sets of resonances. These are apparent for the 32, 121, and P3a methyl protons and are somewhat less apparent for the 134 methyl and 10 methine protons. Surprisingly, the 82 methyl protons exhibit four resonances at the highfield region of the 2D HMQC spectrum. Assignment of the four resonances of 82-H was confirmed using the selectively 13Clabeled BChl ap in which the 82 methyl carbon was efficiently labeled in the 13CH3COONa-containing medium. There is a tendency for the protons in the peripheral groups around pyrrolic rings II and III, that is, 82-H, 81-H, 10-H, and 121-H, to give two resonances, whereas the protons on the opposite side, that J. AM. CHEM. SOC.

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Wang et al. Table 1. Assignments of 1H Chemical Shifts for the BChl a in Reconstituted B820 Subunit position

5 20 10 132 P2 P1 18 7 8 17 134 21 121 32 171 81 172 P4 71 181 a

Figure 4. Downfield regions of 1H NMR spectra. (a) One-dimensional HMQC spectrum of the reconstituted B820 subunit with fully 13C-labeled BChl ap in 5% deuterated OG-phosphate buffer (50 mM, D2O, pH 7.0). (b) Conventional 1H spectrum of natural abundance BChl ap in acetone-d6. (c) One-dimensional HMQC spectrum of natural abundance apopolypeptides in 5% deuterated OG-phosphate buffer (50 mM, D2O, pH 7.0).

is, 21-H, 20-H, and 181-H, appear as single resonances. Two possibilities may be considered to interpret the different behavior. Because the B820 subunit has been determined to exist in the (BChl a)2Rβ form by a small-angle neutron scattering experiment (unpublished result) and other techniques,6 the two BChl a molecules may overlap each other over pyrrolic rings II and III to which the attached protons experience slightly different ring-current effects. In this case, the protons on the other side (rings I and IV) of BChl a macrocycle may be surrounded by polypeptides or detergent molecules in a similar environment. This pigment configuration is supported by the known LH2 crystal structure in which the two BChl a molecules in a subunit are present in a parallel face-to-face conformation with partial overlap over rings III and V.4 Another explanation is that the pigment molecules may adopt a conformation in which the peripheral groups attached to rings II and III are exposed to polypeptides and interact in a different way with the side chains of amino acid residues. In comparison with the BChl ap in acetone, a variety of changes in the chemical shift were observed for the protons of BChl ap in the reconstituted B820 subunit. Relatively large downfield complexation shifts (∆δH > 0.4 ppm) were found for 132-H, 172-H, P1-H, P2-H, and one of the resonances from P3a-H. In contrast, 134-H, 81-H, and two of resonances from 82-H exhibited substantial upfield complexation shifts (∆δH ∼ -0.4 to -1.1 ppm), most of these protons being in the side 1076 J. AM. CHEM. SOC.

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B820

∆δHa

position

B820

∆δHa

8.99 8.18 8.12 7.91 6.44 5.62 4.83 4.37 b 4.27 b 3.85 3.67 3.30 3.46 3.31 3.06 3.21 2.93 2.28 1.43 1.30 2.64 2.02 1.83 1.53 1.53

0.14 -0.26 -0.28 -0.49 0.46 0.48 0.44 0.02 0.02 0.20 -0.29 -0.48 0.02 -0.02 -0.27 0.16 -0.12 -0.13 -1.08 -0.68 0.54 0.11 -0.09 -0.22 -0.14

P3a

2.14 1.81 1.87 1.42 1.42 1.15b 1.15b 1.31 1.13 1.00 1.30 1.29 1.14 1.02 1.15 1.24 0.96 0.59 0.11 0.83 0.72 0.82 0.71 0.87b 0.81b

0.56 0.23 0.35 0.06 0.06 -0.21 -0.17 0.12 0.01 -0.14 0.06 0.04 0.09 -0.03 0.10 0.14 -0.14 -0.51 -0.99 -0.03 -0.14 -0.04 -0.15 0.05 0.00

P15 P7 P11 P5 P9 P13 P14 P8 P10 P12 P6 82

P16 P15a P11a P7a

∆δH ) δΗB820 - δΗ770(acetone-d6). b These assignments are provisional.

chains attached to rings II and V. The result coincides with the observation of two resonances for the corresponding protons and indicates that peripheral groups of rings II, III, and V in the two BChl a molecules are involved in different interactions with either adjacent pigment molecule or side chains of amino acid residues. The large complexation shifts observed for the propionic side chain and the first two groups of phytol chains (P1-H and P2-H) may reflect very different conformations adopted for this portion of the long side chain from that in organic solvents. Similar behavior was also found for BChl c molecules upon dimer formation.19 Dynamic properties revealed by the 1H NMR spectra were striking for both pigment molecules and polypeptides. Protons on the macrocycle of BChl ap exhibited significant signal broadening compared to those of the phytol side chain, as shown in Figure 4. It is worth noting that signals from the highly flexible part of polypeptides can also be resolved even for the natural abundance sample. In Figure 4c, the resonance at 6.85 ppm was assigned as 3-H and 5-H of the phenol ring of tyrosine in the β-polypeptide on the basis of 2D HMQC and COSY experiments where these protons were shown to correlate with neighboring 2-H and 6-H. The unique tyrosine (no tyrosine residue in R-polypeptide) is the second residue from the carboxyl terminus of β-polypeptide and gives rise to sharp signals without labeling. Because the shape and chemical shift of this resonance remained unchanged upon reconstitution, the carboxyl terminal domain of the β-polypeptide is considered to retain a highly mobile motion in the detergent-solubilized B820 subunit solution. 13C NMR Assignment of the BChl a in Reconstituted p B820 Complex. Figure 5 shows the one-dimensional 13C spectrum of BChl ap in the reconstituted B820 subunit using fractionally NaH13CO3-enriched pigments. The overall features are similar to those observed from the corresponding 1H spectra. Signal broadening was remarkable for all carbons of the incorporated BChl ap, and the upfield region was largely dominated by the signals from detergent molecules. Therefore,

Solution NMR of BChl a in B820 Complex

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Figure 5. 13C NMR spectrum of the reconstituted B820 subunit using fractionally 13C-enriched BChl ap with the NaH13CO3-containing medium. The spectrum was acquired in 5% deuterated OG-phosphate buffer (50 mM, D2O, pH 7.0) at 35 °C. Table 2. Assignments of 13C Chemical Shifts of BChl a in the Reconstituted B820 Subunit position

B820

∆δCa

31

203.3 192.0 191.2 174.3 173.4 173.3 168.8 166.9 166.2 162.8 160.8 154.8 152.6 150.3 151.1 144.3 143.3 137.2 130.6 128.6 121.1 111.6 102.8 101.1 97.7 66.8 62.7 56.7b 53.8 52.3b 51.4b

4.0 2.9 2.2 0.8 2.0 0.0 0.1 -0.56 -1.3 2.0 2.1 2.5 1.4 0.0 1.4 1.7 0.8 -0.5 0.2 2.0 1.7 1.6 0.5 1.4 1.1 1.2 1.2 0.9 1.4 1.6 1.7

131 173 133 6 19 14 9 16 1 4 11 P3 2 3 13 12 P2 15 10 5 20 132 P1 8 134 17 18

position

B820

∆δCa

7 P4 P14 P10 P12 P8 P6 P11 P7 32 171 172 81 P15 P13 P9 P5 181 71 P15a P16 P7a P11a P3a 21 121 82

49.2 41.5 41.3 39.1b 39.2 39.1 38.6 34.7 34.1 35.2 32.0 32.0 31.8 30.0 26.7 26.3 26.0 25.6b 25.0 24.7b 24.4 21.9 21.7 18.6 16.1 15.1 13.0 12.4 11.6 10.7

0.7 1.1 1.2 1.0 1.2 1.1 1.3 1.2 0.8 2.1 0.9 0.9 1.1 1.3 1.0 0.8 0.9 2.1 1.7 1.6 1.5 1.8 1.7 2.3 2.4 3.1 2.1 1.5 0.7 -0.2

a ∆δ )δC C b C B820 - δ 770(acetone-d6). These assignments are provisional, see text.

assignment of the upfield carbon resonances had to rely on 2D HMQC and INEPT experiments using the selectively 13Clabeled BChl ap in comparison with the corresponding spectra from apopolypeptides under the same conditions. Using the combined techniques, we have assigned the 13C chemical shifts of BChl ap in the reconstituted B820 subunit, which are shown in Table 2 together with the complexation shifts relative to those in acetone. It should be noted that the 13C chemical shift is

relatively insensitive to the ring-current effect compared to the chemical shift because it is generally governed by mixed effects of paramagnetic shielding, hydrogen bonding, polarity of the solvents, and coordination state, along with the ringcurrent effect.19 In a specific case, change in the 13C chemical shift for an individual carbon may be caused by a single effect. Assignment of the downfield resonances of BChl ap in the reconstituted B820 subunit was made by using the selectively 13C-enriched pigments. Resonances of carbonyl carbons appeared the most downfield, followed by those of carboxyl, quaternary, and aromatic carbons. All carbonyl and carboxyl carbons of BChl ap were found to be highly enriched in the CH313COONa-containing medium with their neighboring carbons unlabeled, leading to a well-resolved spectrum even for the reconstituted complex as shown in Figure 6. The most remarkable features observed from the spectra are the large downfield complexation shifts for the carbonyl carbons and two resonances revealed by the 131 keto carbonyl carbon. The 13C chemical shift of the carbonyl group can be used as a sensitive probe for detecting hydrogen bonding, as we have demonstrated that the formation of a hydrogen bond results in more than a 3 ppm downfield change in the 13C chemical shift for the carbonyl carbon involved.27 The BChl ap molecules in the reconstituted B820 subunit showed a single resonance with a large downfield complexation shift (∆δC ) 4.0 ppm) for the 31-acetyl carbons, indicating strong hydrogen bonding for all these carbonyl groups. The two resonances of 131 keto carbonyl carbons with moderate downfield complexation shifts can be interpreted in terms of different strengths of hydrogen bond interaction. Similar conclusions have been derived from resonance Raman studies. Visschers et al.8 showed that the 31-acetyl groups of all BChl a molecules in the LH1 complex were hydrogen bonded, and about half of the original hydrogen bonds were enhanced upon dissociation of the LH1 complex into the B820 subunits. They also observed two bands corresponding to the frequencies of the 131 keto vibration upon formation of the B820 subunit and proposed that a substantial fraction of the hydrogen bonds were weakened or may even be lost.8 The result of this study is in 1H

(27) Nozawa, T.; Ohtomo, K.; Suzuki, M.; Nakagawa, H.; Shikama, Y.; Konami, H.; Wang, Z.-Y. Photosynth. Res. 1994, 41, 211-223. J. AM. CHEM. SOC.

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All resonances in this region exhibited a downfield change in the chemical shift, and only a few closely spaced resonances interchanged their orders with respect to those in acetone. This may be mainly due to a solvent effect. Several resonances from carbons in pyrrolic rings II, IV, and the phytol group were heavily overlapped even using the selectively 13C-labeled sample; therefore, their assignments remained provisional. Conclusions

Figure 6. Downfield regions of 13C NMR spectra from (a) the reconstituted B820 subunit using 13C-BChl ap labeled with CH313COONa-containing medium, (b) 13C-BChl ap labeled with CH313COONa-containing medium in acetone-d6, and (c) natural abundance apopolypeptides. Both the spectra of the B820 subunit and the apopolypeptides were acquired in 5% deuterated OG-phosphate buffer (50 mM, D2O, pH 7.0) at 35 °C.

good agreement with that of resonance Raman. The equal intensities of the two 13C resonances of 131 keto carbons indicate that the 131 carbonyls involved in different interactions have an exact ratio of 1:1, or in other words, the 131 carbonyl group in one BChl ap molecule within a B820 subunit forms a stronger hydrogen bond than that in another BChl ap molecule. Assignment of quaternary carbons of BChl ap in the B820 complex was mainly made by using the selectively 13C-labeled pigments. Because there is a discrepancy in the assignment of some of these carbons in organic solvents,26 we have reassigned the 13C signals of this region using acetone as a solvent by C-C correlation and long-range H-C correlation techniques as described previously.19 Assignment of the upfield 13C resonances was relatively easy, as chemical shifts of the BChl ap in organic solvent have been well established for these carbons, and H-C correlation can be employed to assist the assignment. There was no peak splitting observed for these carbons upon reconstitution.

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In summary, we have developed a reconstitution method for preparing a highly concentrated and homogeneous solution of the bacterial core light-harvesting complex subunit suitable for NMR measurement. The reconstituted pigment-membrane polypeptide subunit is fully functional and reveals spectroscopic features resembling those from native type. High-resolution NMR spectra have been obtained for the BChl ap molecules in the reconstituted subunit, where fully and selectively 13C-labeled pigments have been incorporated with natural abundance polypeptides in deuterated detergent solution. Molecular motion is significantly reduced for the BChl ap molecules, particularly for the bacteriochlorin macrocycles, upon formation of the complex with polypeptides. The two BChl ap molecules exist in a nonequivalent configuration in the subunit where peripheral groups attached to rings II, III, and IV in the respective molecules are surrounded with different local environments. It is highly likely that the two BChl a molecules adopt a parallel face-to-face conformation with a partial overlap over their pyrrolic rings II, III, and V. In comparison with the BChl ap in acetone, 82, 132, and 134 protons are largely perturbed in the reconstituted subunit, and the propionic and phytol side chain may adopt a very different conformation. Both the 31-acetyl carbonyls in the BChl ap dimer show strong hydrogen bonding with similar strength, whereas 131 keto carbonyl in one BChl ap molecule forms a stronger hydrogen bond than that in another BChl ap molecule. Acknowledgment. The authors thank Prof. M. Nango and Dr. A. Kashiwada for helpful discussion in the early stage of this study. We also thank H. Saito for technical assistance. This work was supported by Grant-in-aid for Scientific Research (12878108 and 12450341) and Scientific Research of Priority Areas: Single-Cell Molecular Technology (11227203), from the Ministry of Education, Science, Sports and Culture, Japan. Supporting Information Available: 1H-13C HMQC spectra

of the B820 subunit reconstituted from various selectively 13Clabeled BChl ap with natural abundance polypeptides along with that of BChl ap in acetone-d6 solution (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. JA0112994